Project Summary Our long-term goal is to advance biomedical science by uncovering the mechanisms of proteins, such as K+ channels that underlie numerous physiologically and pathologically important processes including neuronal activity, blood pressure regulation and cardiac rhythmicity (or arrhythmias). Knowledge of protein mechanisms constitutes the necessary foundation for us to understand physiological and pathophysiological processes, and devise therapeutic strategies to treat diseases. The goal of this proposal is to develop a visible light microscope and use it to quantitatively examine angstrom-scale conformational changes of both soluble and membrane proteins on a physiologically relevant time scale, and to use this technique to uncover, for example, the control mechanisms of Ca2+-dependent K+ channels. Crystallography has produced abundant useful protein structures for understanding protein function. However, full understanding of a protein molecule must include both its spatial and temporal characteristics. We thus need to go beyond describing a protein merely with static pictures and, instead, represent it with a real-time motion picture, i.e., a digital model that simultaneously exhibits both its structural and its kinetic mechanisms with embedded energetic information. However, the required experimental information about protein dynamics is often lacking, due to the absence of relatively general methods for reliably tracking angstrom-scale conformational changes of a protein. Generally, such small changes can be reliably and quantitatively resolved only with such structural techniques as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Conventional light microscopy, on the other hand, may be time-resolved but its spatial resolution remains too low to resolve angstrom-scale changes, despite the landmark achievement with ~20 nm resolution of super-resolution fluorescence microscopy. In the spirit of innovation, we will build a visible-light microscope?with angstrom- and millisecond-scale resolution?to examine multi-state protein-conformational changes in a single protein molecule. We will first develop it using the isolated, soluble control domain of Ca2+-dependent K+ channels as a model, and then extend the technique to membrane proteins, using intact channels. Additionally, in these studies, we will also systematically investigate the energetics and kinetics of the control (or ligand-gating) domain in these channels, information necessary for building a quantitative, mechanistic model that accounts of Ca2+- dependent gating-conformational changes. Such mechanistic inquiry cannot be carried out with existing electrophysiological and microscopic techniques. Success of our proposed study will transform the way we investigate the dynamics/kinetics of protein conformational changes, and accelerate the transition from the current, mostly static approach of structural biology to dynamic structural biology.